High throughput assay for measuring adenovirus replication kinetics

Patent metadata

Field	Value
Publication number	US-11130968-B2
Application number	US-201816109513-A
Country	US
Kind code	B2
Filing date	Aug 22, 2018
Priority date	Feb 23, 2016
Publication date	Sep 28, 2021
Grant date	Sep 28, 2021

Abstract

Recombinant adenovirus genomes that include a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence are described. The recombinant adenovirus genomes and recombinant adenoviruses produced by the disclosed genomes can be used, for example, in high-throughput assays to measure virus replication kinetics. Methods for measuring replication kinetics of a recombinant adenovirus are also described.

First claim

The invention claimed is:

1. A method for measuring replication kinetics of a recombinant adenovirus, comprising:
(i) transfecting cells with the genome of the recombinant adenovirus, or infecting cells with particles of the recombinant adenovirus,
wherein the recombinant adenovirus comprises a recombinant adenovirus genome comprising a heterologous opening reading frame (ORF) encoding a fluorescent protein, and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF, and wherein:
(a) the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k;
(b) the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase;
(c) the endogenous ORF is DNA-binding protein (DBP) and the heterologous ORF is 3′ of DBP;
(d) the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5′ of ADP;
(e) the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k; or
(f) the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2;

(ii) culturing the transfected cells or infected cells for at least two days;
(ii) measuring fluorescence at regular intervals throughout the culture period; and
(iv) calculating log-slope from the fluorescence measurements, thereby measuring replication kinetics of the recombinant adenovirus.

2. The method of claim 1 , wherein the recombinant adenovirus further comprises a second heterologous ORF.

3. The method of claim 1 , wherein replication kinetics of the recombinant adenovirus is measured in a first cell type and a second cell type.

4. The method of claim 3 , wherein the first cell type is a tumor cell and the second cell type is a non-tumor cell.

5. The method of claim 1 , comprising transfecting cells with the genome of the recombinant adenovirus.

6. The method of claim 5 , wherein transfection results in approximately 5-10% of cells transfected.

7. The method of claim 1 , comprising infecting cells with particles of the recombinant adenovirus.

8. The method of claim 7 , wherein the cells are infected with serial dilutions of the recombinant adenovirus particles.

9. The method of claim 1 , wherein the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k.

10. The method of claim 1 , wherein the endogenous ORF is ADP and the heterologous ORF is 5′ of ADP.

11. The method of claim 1 , wherein the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k.

12. The method of claim 1 , wherein the self-cleaving peptide is a 2A peptide or variant thereof.

13. The method of claim 12 , wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof.

14. The method of claim 13 , wherein the amino acid sequence of the self-cleaving peptide is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19.

15. The method of claim 13 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 12-19.

16. The method of claim 1 , wherein the fluorescent protein is a green fluorescent protein (GFP) a yellow fluorescent protein (YFP), a red fluorescent protein (RFP) or a blue fluorescent protein (BFP).

17. The method of claim 16 , wherein the YFP is YPet or the RFP is mCherry.

18. The method of claim 1 , wherein the recombinant adenovirus genome comprises in the 5′ to 3′ direction:
E1B-55K-P2A-YPet;
E1B-55K-P2A-mCherry;
YPet-P2A-(DNA polymerase);
DBP-P2A-YPet;
YPet-P2A-ADP;
E3-14.7k-P2A-YPet;
YPet-P2A-E4-ORF2; or
mCherry-P2A-E4-ORF2.

19. The method of claim 1 , wherein the recombinant adenovirus genome comprises the nucleotide sequence of any one of SEQ ID NOs: 3-7, 9-11, 20 and 21.

20. The method of claim 1 , wherein the recombinant adenovirus genome comprises in the 5′ to 3′ direction YPet-P2A-ADP.

21. The method of claim 20 , wherein the recombinant adenovirus genome comprises the YPet-P2A-ADP sequence of adenovirus CMBT-403, wherein adenovirus CMBT-403 has a genome of SEQ ID NO: 7.

Assignees

• Salk Inst For Biological Studi

Inventors

• O'Shea Clodagh
• Partlo William
• Powers Colin

Classifications

• C07K14/005 Primary

  from viruses · CPC title

• C12N2710/10331

  Uses of virus other than therapeutic or vaccine, e.g. disinfectant · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C07K2319/01

  containing a localisation/targetting motif · CPC title